Histochemistry (1992) 98 : 311-315

Histochemistry © Springer-Verlag 1992

The intramucosal distribution of gastric alcohol dehydrogenase and aldehyde dehydrogenase activity in rats I.P. Maly 1, M. Arnold 1, K. Krieger 1, M. Zalewska 2, D. Sasse 1 1 Anatomisches Institut der Universitfit Basel, Pestalozzistrasse 20, CH-4056 Basel, Switzerland 2 Clinic of Infectious Disease, Medical University of Wroclaw, PL-53~416 Wroclaw, Poland Accepted: 28 August 1992

Abstract. Using qualitative and microquantitative histochemical techniques, alcohol dehydrogenase and aldehyde dehydrogenase activity was studied in the gastric mucosa of male and female rats. Alcohol dehydrogenase was demonstrated by staining reactions with maximum activity in surface and neck cells and with clearly weaker activity also in parietal cells. Aldehyde dehydrogenase could be detected in surface and neck cells, and also to a comparable degree in the parietal cells. Quantitative analyses of microdissected samples yielded high values for alcohol dehydrogenase activity exclusively in the superficial part of the gastric mucosa, whereas low-Kin aldehyde dehydrogenase activity showed a decreasing gradient from the surface to the deeper parts of the mucosa. Sex differences could not be confirmed.

Introduction In earlier studies alcohol dehydrogenase and aldehyde dehydrogenase activity was demonstrated to be heterotopically distributed in the liver parenchyma. These enzymes, which are essentially involved in ethanol degradation possess not only a typical intra-acinar distribution pattern but also exhibit activity that varies according to sex in the perivenous zones in the human and rat liver (Maly and Sasse 1985, 1987a, b, 1988, 1991). These results gave support to the hypothesis that sex-specific enzymatic heterogeneity in the liver may be the cause of the earlier and more severe development of alcoholic damage in women than in men. Recent publications, however, emphasize a further important role of ethanol degrading enzymes in the wall of the digestive tract, mainly of the stomach (Lamboeuf et al. 1981; Julkunen et al. 1985). These enzymes probably play the decisive Dedicated to Professor Dr. K.S. Ludwig on the occasion of his 70th birthday Correspondence to: D. Sasse

role in the so-called first-pass metabolism of ethanol. It has been claimed that alcohol dehydrogenase in the gastric mucosa shows sex-specific activity, which might, perhaps additionally, contribute to the different susceptibility of the liver to alcoholic beverages (Frezza et al. 1990). The studies cited above on gastric enzyme activity were carried out in homogenates of bioptic material so that exact localization of the enzymes was not possible. Earlier studies on the topochemistry of alcohol dehydrogenase were either carried out with as yet insufficient techniques (Ferguson 1965), or modern immunohistochemical methods were used without taking into consideration possible sex differences (Pestalozzi et al. 1983). Aldehyde dehydrogenase activity has never before been demonstrated. In previous studies the technique for the demonstration of alcohol dehydrogenase activity has been improved by using viscous medium and bY application of 300 m M Tris as an aldehyde trapping agent (Maly and Sasse 1985, 1987b). Furthermore, a histochemical method for the demonstration of aldehyde dehydrogenase activity is described in the present study for the first time. The microquantitative methods in the nanogram range make the interpretation of the staining reactions fully objective. This must contribute to the resolution of the question whether or not the alcohol degrading enzymes show sex specific differences in the gastric mucosa as they do in the liver acinus.

Materials and methods Ten adult Wistar rats (five males weighing 250.0+__3.0g; five females weighing 189.0± 5.0 g) were obtained from Mad6rin, Filllinsdorf, Switzerland. The animals were kept in separate cages at a constant room temperature of 21° C with a 12 h light/dark cycle (7.00 a.m.-7.00 p.m.). They had free access to water and Kliba standard diet. The rats were stunned and sacrificed by decapitation between 9.00 and 11.00 a.m. and their stomachs were immediately removed. In order to obtain a flat mucosal surface, the stomach was pinned onto a cork plate covered with a thin polyester foil.

312 The pins were inserted along the greater and the lesser curvature and along the borderline between the proventricular and glandular regions. Whereas all other parts were discarded, the area of fundic glands attached to the cork plate was preserved and rinsed for a short time with 60 mM potassium phosphate, pH 7.4 at room temperature. In order to protect the epithelial cells on the surface from drying and mechanical destruction, the mucosa was covered with a 3 to 4-mm-thick layer of a homogenized meat paste, analogous to the brain paste (Lowry and Passonneau, 1972). After these procedures, which lasted no longer than 3 min, the tissue on the cork plate was frozen in liquid nitrogen. The pins and the cork plate with the gel bond foil were then removed in a cryostat at - 2 0 ° C. The frozen tissue was cut in half with a razor blade perpendicularly to the long axis of the stomach. The tissue was stored in air-tight tubes at - 80° C until required. Fifteen cross-sections (16 btm) of the area of fundic glands from each rat were cut at a cryostat temperature of - 2 0 ° C. Special care was taken to ensure that the protective meat layer remained attached to the mucosal surface. Five sections were used for the histochemical demonstration of enzyme activity. The other sections of unfixed tissue were lyophilized in a vacuum (666 mPa) at - 4 0 ° C for about 24 h. For qualitative histochemistry, cryostat sections thawed on coverslips were dried for 5 rain at 37° C. Alcohol dehydrogenase activity was demonstrated in accordance with a method earlier described (Maly and Sasse 1987b). Aldehyde dehydrogenase activity was demonstrated after incubation in the following medium: 60 mM sodium phosphate buffer, pH 7.4; 30% polyvinylalcohol, I mM EDTA, 1 mM amytal, 0.5 mM methylpyrazole, I mM nicotinamide adenine dinucleotide (NAD), 5 mM nitro blue tetrazolium (NBT), 5 mM acetaldehyde and 0.33 mM phenazine methosulphate (PMS). Controls were incubated in media without substrates. After incubation all sections were postfixed in a solution of 4% formaldehyde, 136 mM CaCl2 and 10% polyvinylpyrrolidone and mounted in glycerin jelly. Using the stained sections as guides, an area was chosen from the lyophilized tissue where the fundic glands were oriented longitudinally. From each stomach, 6-7 strips of the mucosa were microdissected. Each strip contained 3-4 fundic glands. The protective meat layer and the mucus covering the gastric epithelium, as well as the submucosal tissue at the other end of the strip, were removed, The remaining strip was then subdivided into 5 samples of 50-150 ng, as weighed on a quartz fibre balance. Microquantitative determination of the alcohol dehydrogenase activity was carried out as described by Maly and Sasse (1985), but with a substrate concentration of 1 M ethanol, low K,~ aldehyde dehydrogenase activity with 80 gm acetaldehyde as substrate was determined using the method described by Maly and Sasse (1987a). The amount of specifically produced NADH was measured luminometrically. For the demonstration of the distribution pattern of enzyme activity in the gastric mucosa, the weight of the subsamples was related to the weight of the whole strip. According to its weight, each subsample represented a certain part of the total length of the strip of mucosa. Thus it was possible to plot the enzyme activity (y-values) against the relative size and location of the corresponding tissue samples (x-values) of one microdissected strip, or of all the strips from one animal. Mean values and standard deviations were calculated for 200 points on the abscissa, which together represented the total height of the mucosa.

Results

Histochemistry Alcohol dehydrogenase (ADH). After incubation for the d e m o n s t r a t i o n o f A D H activity, a deeply stained b a n d m a r k e d the luminal part o f the mucosa. At higher magnification the reaction p r o d u c t was localized in the basal

cytoplasmic regions o f the surface cells and the neck cells. In the deeper parts o f the glands a m o d e r a t e reaction was visible in the parietal cells, but the staining intensity sometimes hardly differed f r o m the backg r o u n d reaction which was also present in controls. The chief cells showed a negative staining reaction. Sex specific differences were n o t discernible (Fig. 1).

Aldehyde dehydrogenase (ALDH).The reaction p r o d u c t occurred maximally in the u p p e r part o f the mucosa, but was also localized in the deeper parts o f the gastric glands, where the parietal cells were specifically stained. Higher magnification revealed that surface and neck cells reacted with their basal cytoplasmic regions, whereas in parietal cells the total cytoplasm was stained. Chief cells were negative. This heterotopic distribution pattern was similar in b o t h sexes (Fig. 2).

Microchemistry ADH. The microquantitative distribution pattern exhibited highest A D H activitiy in the superficial tenth o f the m u c o s a with values o f 14.18_+,2.28 btmol/min per g in males and o f 13.91 +_3.06 p m o l / m i n per g in females. This m a x i m u m activity decreased drastically in the direction o f the deeper tissue regions, so that in the second tenth there was only one-half o f the initial activity. After the first third o f the tunica m u c o s a the values were less than 1, and in the deepest third o f the mucosa, A D H activity was no longer detectable. The total values o f A D H activity (males, 2.66+_0.75; females, 2.60_+ 0.74 btmol/min per g) and the distribution pattern did n o t indicate any sex difference (Figs. 3, 4). ALDH (low-Km). A L D H activity showed m a x i m u m values in the superficial p a r t o f the m u c o s a with values o f 5.84+0.51 g m o l / m i n per g in males and 5.65_+ 3.17 g m o l / m i n per g in females. A gradual decrease o f activity could be observed in the direction o f the deeper parts, where in the last quarter the lowest values o f 0.79 _+0.17 ktmol/min per g in males and 0.84_+ 0.17 g m o l / m i n per g in females were attained. Neither the total activity (males, 2.55_+0.38; females, 2 . 5 4 + 0.41 p,m o l / m i n per g) n o r the distribution pattern indicated any sex difference (Figs. 5, 6).

Discussion Microquantitative determinations o f enzyme activities in microdissected tissue samples not only improve the objectivity o f histochemical staining, b u t also serve as a specificity control o f the qualitative histochemical reaction. O n the other hand, the histochemical demonstration o f enzyme activity by staining techniques allows the quantified enzyme activity to be localized accurately, which is indispensable because measurements o f tissue samples even o f only a b o u t 100 ng comprise different cell types. By c o m b i n i n g the qualitative and quantitative histo-

313

tivity. Reaction product occurs mainly in the surface and neck cells of the gastric mucosa, with a moderate reaction of the parietal cells, x 190

Fig. 2. Histochemical demonstration of aldehyde dehydrogenase activity. The reaction product occurs in the surface and neck cells; in the deeper parts of the mucosa the parietal cells are also markedly stained, x 190

chemical results it becomes evident that A D H has a clear-cut m a x i m u m activity in the surface and neck cells of the gastric mucosa. Parietal cells contribute to only a minor part of this total activity, and chief cells appear to be practically negative. The distribution pattern of A D H activity in the stomach o f the rat described here parallels the immunohistochemical results obtained from A D H protein in the gastric mucosa of m a n (Pestalozzi et al. 1983). Quantitatively, A D H activity of the apical tenth of the gastric mucosa shows a ratio to the second and third

tenths of 4:2: 1. This means that the first step of enzymatic ethanol degradation is mainly restricted to the upper third of the mucous membrane, and that the deeper parts must be considered to be of minor functional importance. These results further support earlier reports that the enzymatic degradation of ethanol is not restricted to the liver, significant fractions of oral doses of alcohol being oxidized in the gastrointestinal tract, particularly in the stomach (Julkunen et al. 1985; Caballeria et al. 1987). As a consequence, acetaldehyde and acetate appear

Fig. 1. Histochemical demonstration of alcohol dehydrogenase ac-

314 in the veins draining the stomach following the intragastric administration of ethanol. Julia et al. (1987) reported that, in the rat, ADH-1 is the only isoenzyme that is responsible for this initial step of gastric ethanol metabolism. The A D H - 1 isoenzyme shows a high Km for ethanol in the m o l a r range, and is therefore clearly different from the liver A D H - 3 isoenzyme. A similar result was recently described by Yin et al. (1990) for h u m a n gastric A D H . Our microchemical results on A D H activity were achieved by using a substrate concentration of 1 M ethanol.

The concentration of A D H activity in the upper part of the mucosa could be relevant for the interpretation of alterations in the first-pass ethanol metabolism under the conditions of alcohol consumption. Kvietys et al. (1990) found that after 40 min of perfusion with 20% ethanol, nearly all of the surface cells are lucent or even detached from the mucosa. Thus, the intragastric administration of ethanol leads to injuries of exactly those cells which, according to the results presented here, are mainly involved in ethanol oxidation. Consequently, the observations of Chey (1972) that in rats chronically fed

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~urface basal" basal ~udace basal Fig. 5. Microquantitative determination of low-Kin aldehyde dehyFig. 3. Microquantitative distribution of alcohol dehydrogenase acdrogenase activity in the gastric mucosa of male rats. Activity was tivity in the gastric mucosa of male rats. Activity was calculated calculated from 150microdissected samples of 30 surface/basal from 160 microdissected samples of 32 surface/basal strips of tisstrips of tissue. Data are of mean values (median line) and standard sue. Data are of mean values (median line) and standard deviations deviations Fig. 4. Microquantitative distribution of alcohol dehydrogenase acFig. 6. Microquantitative determination of low-Km aldehyde dehytivity in the gastric mucosa of female rats. Activity was calculated drogenase activity in the gastric mucosa of female rats. Activity from 170 microdissected samples of 34 surface/basal strips of tiswas calculated from 165 microdissected samples of 33 surface/basal sue. Data are of mean values (median line) and standard deviations strips of tissue. Data are of mean values (median line) and standard deviations

315 on an alcoholic diet there is a decrease in gastric A D H activity, can be interpreted as the result of mucosal injury. Seitz et al. (1983) found that chronic alcohol ingestion results in a significant increase in D N A synthesis in the gastric mucosa. This was interpreted as the consequence of the cellular hyper-regenerative response, which is accompanied by an increase in the parietal cell mass (Lillibridge et al. 1973), as seen also in dogs. It is feasible that, following injury to the mucosal surface, the parietal cells on the more protected sites of the gastric glands try to compensate for the loss of A D H activity. The appearance of acetate in the effluent venous blood of the stomach (Julkunen et al. 1985) suggests that there is further degradation of ethanol in the gastric mucosa. The A L D H distribution profiles described here indicate that the surface and neck cells also play a major role in acetaldehyde oxidation. As a consequence, the upper part of the gastric mucosa is mainly involved in acetaldehyde oxidation. However, the A L D H distribution profiles show that the deeper parts of the mucosa also are capable of the degradation of acetaldehyde. The decreasing gradient of mucosal A L D H activity corresponds to the decrease in the number of parietal cells towards the deeper parts of the gastric glands. Therefore acetaldehyde produced mainly in the upper third of the mucous membrane, can at least be partly oxidized. Frezza et al. (1990) reported sex dependent differences of A D H activity in men and women, with a male/ female ratio of 1.68:1. The authors concluded that because of the lower gastric first-pass metabolism in the female, the bioavailability of ethanol is much greater in women than in men, a phenomenon which should contribute to the higher susceptibility to liver injury found in women compared with men. The results presented here give no hint of sex differences in the gastric mucosa of rats, although sex dependent distribution patterns of A D H and low-Kin A L D H activity are clearly evident in the liver (Maly and Sasse 1985, 1987a). These different enzymatic localizations are modified by sex hormones (Maly and Sasse 1987b). In a recent study (Maly and Sasse 1991), the sex differences observed in rats could also be confirmed for the enzymes A D H and A L D H in the liver parenchyma of men and women. Further studies using our methodology should elucidate the discrepancy between our observations in rat stomach and the reports by others on sex-specific differences of A D H in h u m a n gastric mucosa.

Acknowledgements. The stay of M.Z. was financially supported by the Schweizerischer Nationalfonds (SoforthiIfemassnahmen Osteurop/iische Staaten). We thank D. Mfiller for typing the manuscript and Dr. F. Steel for his help with the English text.

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